Bottom Line:
Transformants carrying tsf or PSEEN0851 also showed an increased resistance to cyclohexane and other organic solvents compared with the control.Of these three genes, mmsB exhibited the most prominent effect on increasing OST of E. coli.Less oxidation product of cyclohexane was detected because mmsB transformants might help keep a lower intracellular cyclohexane level.

ABSTRACTOrganic solvents are toxic to most microorganisms. However, some organic-solvent-tolerant (OST) bacteria tolerate the destructive effects of organic solvent through various accommodative mechanisms. In this work, we developed an OST adapted strain Pseudomonas putida JUCT1 that could grow in the presence of 60% (v/v) cyclohexane. Two-dimensional gel electrophoresis was used to compare and analyze the total cellular protein of P. putida JUCT1 growing with or without 60% (v/v) cyclohexane. Under different solvent conditions, five high-abundance protein spots whose intensity values show over 60% discrepancies were identified by MALDI-TOF/TOF spectra. Specifically, they are arginine deiminase, carbon-nitrogen hydrolase family putative hydrolase, 3-hydroxyisobutyrate dehydrogenase, protein chain elongation factor EF-Ts, and isochorismatase superfamily hydrolase. The corresponding genes of the latter three proteins, mmsB, tsf, and PSEEN0851, were separately expressed in Escherichia coli to evaluate their effect on OST properties of the host strain. In the presence of 4% (v/v) cyclohexane, E. coli harboring mmsB could grow to 1.70 OD(660), whereas cell growth of E. coli JM109 (the control) was completely inhibited by 2% (v/v) cyclohexane. Transformants carrying tsf or PSEEN0851 also showed an increased resistance to cyclohexane and other organic solvents compared with the control. Of these three genes, mmsB exhibited the most prominent effect on increasing OST of E. coli. Less oxidation product of cyclohexane was detected because mmsB transformants might help keep a lower intracellular cyclohexane level. This study demonstrates a feasible approach for elucidating OST mechanisms of microorganisms, and provides molecular basis to construct organic-solvent-tolerant strains for industrial applications.

Mentions:
In order to validate the OST-related function of above three genes, recombinant plasmids harboring mmsB, tsf, and PSEEN0851 were constructed and over-expressed in E. coli JM109 individually (Fig. 3). The recombinant strains were cultured in LBGMg medium till 0.2 OD660 when cyclohexane was added. As shown in Fig. 4, all 3 transformants exhibited higher cyclohexane tolerance than the control (E. coli JM109 carrying empty pQE-80L) when grown in the presence of 4% (v/v) cyclohexane. Especially for recombinant strain over-expressing mmsB gene, a stunning cell density of 1.70 OD660 was reached after 8 h. The expression of PSEEN0851 and tsf also contributed to the higher OST of recombinant E. coli, rendering 0.58 and 0.25 increase in OD660 respectively. For the control strain, however, no appreciable growth was observed after the addition of cyclohexane.

Mentions:
In order to validate the OST-related function of above three genes, recombinant plasmids harboring mmsB, tsf, and PSEEN0851 were constructed and over-expressed in E. coli JM109 individually (Fig. 3). The recombinant strains were cultured in LBGMg medium till 0.2 OD660 when cyclohexane was added. As shown in Fig. 4, all 3 transformants exhibited higher cyclohexane tolerance than the control (E. coli JM109 carrying empty pQE-80L) when grown in the presence of 4% (v/v) cyclohexane. Especially for recombinant strain over-expressing mmsB gene, a stunning cell density of 1.70 OD660 was reached after 8 h. The expression of PSEEN0851 and tsf also contributed to the higher OST of recombinant E. coli, rendering 0.58 and 0.25 increase in OD660 respectively. For the control strain, however, no appreciable growth was observed after the addition of cyclohexane.

Bottom Line:
Transformants carrying tsf or PSEEN0851 also showed an increased resistance to cyclohexane and other organic solvents compared with the control.Of these three genes, mmsB exhibited the most prominent effect on increasing OST of E. coli.Less oxidation product of cyclohexane was detected because mmsB transformants might help keep a lower intracellular cyclohexane level.

ABSTRACTOrganic solvents are toxic to most microorganisms. However, some organic-solvent-tolerant (OST) bacteria tolerate the destructive effects of organic solvent through various accommodative mechanisms. In this work, we developed an OST adapted strain Pseudomonas putida JUCT1 that could grow in the presence of 60% (v/v) cyclohexane. Two-dimensional gel electrophoresis was used to compare and analyze the total cellular protein of P. putida JUCT1 growing with or without 60% (v/v) cyclohexane. Under different solvent conditions, five high-abundance protein spots whose intensity values show over 60% discrepancies were identified by MALDI-TOF/TOF spectra. Specifically, they are arginine deiminase, carbon-nitrogen hydrolase family putative hydrolase, 3-hydroxyisobutyrate dehydrogenase, protein chain elongation factor EF-Ts, and isochorismatase superfamily hydrolase. The corresponding genes of the latter three proteins, mmsB, tsf, and PSEEN0851, were separately expressed in Escherichia coli to evaluate their effect on OST properties of the host strain. In the presence of 4% (v/v) cyclohexane, E. coli harboring mmsB could grow to 1.70 OD(660), whereas cell growth of E. coli JM109 (the control) was completely inhibited by 2% (v/v) cyclohexane. Transformants carrying tsf or PSEEN0851 also showed an increased resistance to cyclohexane and other organic solvents compared with the control. Of these three genes, mmsB exhibited the most prominent effect on increasing OST of E. coli. Less oxidation product of cyclohexane was detected because mmsB transformants might help keep a lower intracellular cyclohexane level. This study demonstrates a feasible approach for elucidating OST mechanisms of microorganisms, and provides molecular basis to construct organic-solvent-tolerant strains for industrial applications.